昆蟲基因組DNA提取試劑盒
Insect
Genomic DNA Isolation Kit
● 試劑盒內容及保存：
|
試劑盒組成
|
RTG2408
（50次）
|
貯存方式
|
|
緩沖液IL
|
22
ml
|
常溫
|
|
緩沖液IB（濃縮液）
|
26
ml
|
常溫
|
|
緩沖液LF
|
16
ml
|
常溫
|
|
DNA
Wash Buffer (濃縮液)
|
65
ml
|
常溫
|
|
緩沖液WB(濃縮液)
|
30
ml
|
常溫
|
|
洗脫緩沖液EB
|
15
ml
|
常溫
|
|
蛋白酶K
|
1.05
ml
|
-20℃
|
|
RNase
A
|
220
μl
|
-20℃
|
|
吸附柱CG（含收集管）
|
50套
|
常溫
|
|
說明書
|
1份
|
|
● 儲存條件和效期：
室溫保存，12個月內有效。緩沖液IL與緩沖液 IB可能有沉淀產生，37℃水浴溶解后即可。RNase
A和蛋白酶K常溫運輸，-20℃保存。
● 產品簡介：
該試劑盒采用獨特的裂解液能夠有效除去多糖多酚等，能夠從昆蟲、軟體動物、節(jié)肢動物、蛔蟲等樣品中提取DNA，保存在醇類的樣品也適用于本試劑盒的提取。一次操作可以處理小于50mg組織，樣品經裂解液消化，氯仿分離除去大部分的多糖多酚，再經分離柱進一步純化，便可得到高純度的DNA。所得的DNA可以用于PCR，Southern雜交，酶切消化等實驗。
● 準備工作：
1. 準備65℃水??；無水乙醇；制冰機；1.5ml離心管；2ml離心管
2. 按照標簽所示在緩沖液IB中加入異丙醇，在DNA Wash Buffer和緩沖液WB中加入無水乙醇，混勻后蓋緊瓶蓋后常溫貯存?zhèn)溆谩?
3. 每次使用前請檢查緩沖液IL，緩沖液IB是否有沉淀生成，如果出現(xiàn)沉淀，37℃溫浴至沉淀溶解后再使用。
● 標準操作步驟：
如非指出，所有離心步驟均為使用臺式離心機在常溫下離心。
1. 液氮充分研磨樣品。
2. 收集研磨成粉末的50
mg樣品，置于1.5ml離心管中。
3. 加入350 μl65℃預熱的緩沖液 IL，并加入20 μl 蛋白酶K劇烈地漩渦振蕩，確保所有的組織團都分散均勻。
4.65℃水浴20-30min。水浴期間顛倒樣品數(shù)次。
5. 加入300 μl緩沖液LF，充分混勻，12,000 rpm (~13,400×g )
離心5分鐘。
6. 小心地把上清液吸至另一新的1.5ml離心管中。注意確保不要打散沉淀團或把組織碎片也一起轉移。
7. 加入4 μl
RNase A，渦旋混勻。常溫放置2min。
8. 加入等體積的緩沖液
IB（請確保已經按照標簽加入異丙醇），充分混勻。如：向300μl上清中加入300 μl 緩沖液 IB。
9. 把上述混勻的液體轉移到吸附柱CG上。10,000×g離心1
min以結合DNA，棄去濾出液體。純化柱最大容量為750 μl,如果混合液大于750 μl,請分兩次過柱。
10. 將吸附柱重新套回收集管中，加入500μl
緩沖液WB（請確保已經按照標簽加入無水乙醇）至柱子中，10,000×g離心1min,倒棄流出液；
11. 將吸附柱重新套回收集管中，加入600μl
DNA Wash Buffer（請確保已經按照標簽加入無水乙醇）至柱子中，10,000×g離心1min,倒棄流出液；
注意：DNA
Wash Buffer使用前須按要求用無水乙醇稀釋。如果放入冰箱中，使用前須恢復到室溫。
12.再加入600 μl
DNA Wash Buffer（請確保已經按照標簽加入無水乙醇）至柱子中，8,000×g離心1min,棄去流出液；
13. 將吸附柱重新套回2ml收集管中，最大轉速（>13000×g）離心空結合柱1min以干燥柱子的基質；這一步對下面的洗脫步驟至關重要。
14. 將柱子置于1.5
ml滅菌離心管，加入50-150 μl 65℃預熱的洗脫緩沖液EB至柱子的膜中央。常溫靜置5 min；
15. 常溫下，離心(>13000g)1min，以洗脫DNA。保留含DNA的流出液。將DNA儲于-20℃。
● DNA濃度及純度檢測：
基因組DNA片段的大小與樣品保存時間、操作過程中的剪切力等因素有關。提取的DNA片段可用瓊脂糖凝膠電泳和紫外分光光度計檢測濃度與純度?？膳渲?.8-1.0％瓊脂糖凝膠，使用λ/HindIII判斷基因組的大小，完整的基因組大小應在23 kb以上。使用分光光度計檢測時， OD260/OD280比值應為1.7–1.9之間，如果洗脫時不使用洗脫緩沖液，而使用去離子水洗脫，比值可能偏低，但并不表明DNA純度不高。
|
常見問題
|
可能原因
|
建議
|
|
堵柱子
|
轉移裂解上清時，轉移了沉淀
|
按說明書操作，緩沖液LF分離后，確保不轉移到沉淀
|
|
樣品太粘稠
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樣品量別超過說明書上所說的，或者增加緩沖液IB的用量。
|
|
低濃度的DNA量
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樣品的破壁方式不對
|
不論新鮮還是干燥樣品，在加入緩沖液IL之前必須用適當方式的研磨成粉末。
|
|
樣品的裂解效果不好
|
減少樣品量，或者增加緩沖液IL的用量。
|
|
DNA殘留在柱子上
|
增加洗脫液EB的用量，并在離心洗脫前將洗脫液EB65℃孵育5min。
|
|
DNA洗滌不當
|
DNA
Wash Buffer按說明書用無水乙醇稀釋。
|
|
下游應用不好
|
提取的DNA含鹽量高
|
DNA
Wash Buffer必須按要求用無水乙醇稀釋，必須常溫放置。
|
|
提取的DNA含有乙醇
|
洗脫前，必須最高轉速空甩柱子1 min。
|
核酸提取產品發(fā)表文章
1. [2008 IF=1.749] Development of a sequence-characterized
ampli?ed region marker for diagnosis of dwarf bunt of wheat and detection of
Tilletia controversa Kuhn.
Author: J.H. Liu, L. Gao, T.G. Liu and W.Q. Chen
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: Letters in Applied Microbiology 2009,49,235-240
Institution：Institute of
Plant Protection ,Chinese Academy of Agricultural Sciences
Paper link： https://doi.org/10.1111/j.1472-765X.2009.02645.x
2. [2009 IF=2.435] Characterization of three new S-alleles
and development of an S-allele-specific PCR system for rapidly identifying the
S-genotype in apple cultivars.
Author: Shenshan Long, Maofu Li, Zhenhai Han, Kun Wang, Tianzhong Li
Product: RTP2201瓊脂糖凝膠回收試劑盒
Journal: Tree Genetics & Genomes (2010)
6:161–168
Institution：China
Agricultural University
Paper link： https://link.springer.com/article/10.1007/s11295-009-0237-6
3. [2010 IF=0.921] An ISSR-based Approach for the Molecular
Detection and Diagnosis of Dwarf Bunt of Wheat, Caused by Tilletia controversa
Kuhn.
Author: Li Gao,Wanquan Chen and Taiguo Liu
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: J Phytopathol 159:155–158 (2011)
Institution：State Key
Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant
Protection, CAAS
Paper link：https://onlinelibrary.wiley.com/doi/10.1111/j.1439-0434.2010.01735.x
4. [2010 IF=1.359] Curing the Plasmid pXO2 from Bacillus
anthracis A16 Using Plasmid Incompatibility.
Author: Huagui Wang, Xiankai Liu, Erling Feng, Li Zhu, Dongshu Wang, Xiangru Liao,
Hengliang Wang
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: Curr Microbiol (2011) 62:703–709
Institution：State Key
Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology
Paper link：https://link.springer.com/article/10.1007/s00284-010-9767-2
5. [2010 IF=1.993] Computation-assisted SiteFinding-PCR for
isolating flanking sequence tags in rice
Author: Hongru Wang, Jun Fang, Chengzheng Liang, Minghui He, Qiye Li, and
Chengcai Chu
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: BioTechniques Vol. 51 | No. 6 | 2011
Institution：Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences
Paper link：https://pubmed.ncbi.nlm.nih.gov/22150334/
6. [2012 IF=0.903] T Polymorphisms in major
histocompatibility complex class IIa genes are associated with resistance to
infectious hematopoietic necrosis in rainbow trout, Oncorhynchus mykiss
(Walbaum, 1792).
Author: Z. Liu, D.-D. Hu, S.-J. Shao, J.-Q. Huang, J.-F. Wang and J. Yang
Product: RTP2202 PCR產物純化試劑盒
Journal: J. Appl. Ichthyol. 29 (2013), 1234–1240
Institution：College of
Animal Science and Technology, Gansu Agricultural University
Paper link：https://onlinelibrary.wiley.com/doi/full/10.1111/jai.12326
7. [2012 IF=1.958] Cloning, bioinformatics and the enzyme
activity analyses of a phenylalanine ammonia-lyase gene involved in dragon’s blood biosynthesis in Dracaena
cambodiana.
Author: Xing-Hong Wang，Changhe Zhang
Product: RTP2202 PCR產物純化試劑盒
Journal: Mol Biol Rep (2013) 40:97–107
Institution：Yunnan Institute
of Microbiology, Yunnan University
Paper link：https://link.springer.com/article/10.1007/s11033-012-2032-y
8. [2013 IF=1.687] Tobacco Arabinogalactan Protein NtEPc Can
Promote Banana (Musa AAA) Somatic Embryogenesis.
Author: H. Shu & L. Xu & Z. Li & J. Li & Z. Jin & S. Chang
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: Appl Biochem Biotechnol (2014)
174:2818–2826
Institution：Haikou
Experimental Station, Chinese Academy of Tropical Agricultural Sciences
Paper link：https://link.springer.com/article/10.1007/s12010-014-1228-0
9. [2015 IF=1.32] Association between MHC II beta chain gene
polymorphisms and resistance to infectious haematopoietic necrosis virus in
rainbow trout (Oncorhynchus mykiss, Walbaum, 1792).
Author: Juan Yang, Zhe Liu, Hai-Na Shi, Jiu-Pan Zhang, Jian-Fu Wang, Jin-Qiang
Huang & Yu-Jun Kang
Product: RTP2202 PCR產物純化試劑盒
Journal: Aquaculture Research, 2016, 47,
570–578
Institution：College of
Animal Science and Technology, Gansu Agricultural University
Paper link： https://onlinelibrary.wiley.com/doi/10.1111/are.12516
10. [2015 IF=] A weird DNA band in PCR and its cause.
Author: Chang Shenghe, Sun Wei, Zhou Zhaoxi, Li Jingyang, Dai Minjie, Shu Haiyan
Product: RTP2102 普通質粒小提試劑盒
Journal: Journal of Plant Science & Molecular
Breeding Volume 5 Article 2 2016
Institution：Haikou
experimental station, Chinese Academy of Tropical Agricultural Sciences
Paper link：
11. [2017 IF=4.076] Application of Real-Time Quantitative PCR
to Detect Mink Circovirus in Naturally and Experimentally Infected Minks.
Author: Xingyang Cui, Yunjia Shi, Lili Zhao, Shanshan Gu, Chengwei Wei, Yan
Yang,Shanshan Wen, Hongyan Chen and Junwei Ge
Product: RTP2102 普通質粒小提試劑盒
Journal: Fronties in Microbiology May 2018 | Volume 9 | Article 937
Institution：College of
Veterinary Medicine, Northeast Agricultural University
Paper link：https://pubmed.ncbi.nlm.nih.gov/29867846
12. [2017 IF=3.974] In vitro and in vivo toxic e?ects and in?ammatory responses induced by carboxylated
black carbon-lead complex exposure.
Author: Shuanglin Jiang,, Mengting Shang,Kui Mu，Nan Jiang，Haiyan Wen，Rong Wang，Hai Wu，Wenyong Li
Product: RTG2402 動物/細胞基因組DNA提取試劑盒
Journal: Ecotoxicology and Environmental
Safety 165 (2018)
484–494
Institution：Key Laboratory
of Embryo Development and Reproductive Regulation of Anhui Province, Fuyang
Normal University
Paper link：http://www.sciencedirect.com/science/article/pii/S0147651318309011
13. [2018 IF=8.063] ATF4 destabilizes RET through nonclassical
GRP78 inhibition to enhance chemosensitivity to bortezomib in human
osteosarcoma
Author: Jie Luo,# Yuanzheng Xia,# Yong Yin, Jun Luo, Mingming Liu, Hao Zhang,
Chao Zhang, Yucheng Zhao, Lei Yang, and Lingyi Kong
Product: RTP2101 高純質粒小提試劑盒
Journal: Theranostics 2019, Vol. 9, Issue 21
Institution：School of
Traditional Chinese Pharmacy, China Pharmaceutical University
Paper link：https://pubmed.ncbi.nlm.nih.gov/31534554
14. [2020 IF=1.857] Characterization and Developmental
Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus
terrestris (Hymenoptera: Apidae).
Author: Yakai Tian, Yingping Qu,Kun Dong,Shaoyu He,Wu Jie, and Jiaxing Huang
Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: Journal of Insect Science (2021) 21(5): 13; 1–8
Institution：Institute of
Apicultural Research, Chinese Academy of Agricultural Sciences
Paper link：https://doi.org/10.1093/jisesa/ieab078
15. [2020 IF=1.336] Isopentenyl Diphosphate Isomerase (IPI)
Gene Silencing Negatively Afects Patchouli Alcohol Biosynthesis
in Pogostemon cablin
Author: Wuping Yan, Yuzhang Yang, Yougen Wu, Jing Yu, Junfeng Zhang,
Dongmei Yang, Zeeshan Ul Haq Muhammad
Product: RTP2102 普通質粒小提試劑盒
Journal: Plant Molecular Biology Reporter Published 27 January 2021
Institution：College
of Horticulture, Hainan University
Paper link：https://link.springer.com/article/10.1007/s11105-020-01269-0
16. [2021 IF=6.064] Genome resequencing and transcriptome
analysis reveal the molecular mechanism of albinism in Cordyceps militaris.
Author: Ying Zhao, YuDong Liu, Xun Chen and Jun
Xiao
Product: RTG2407 真菌基因組DNA提取試劑盒
Journal: Fronties in
Microbiology. Published 11 April 2023
Institution： Institute of Edible Fungi, Liaoning
Academy of Agricultural Sciences
Paper link：https://www.frontiersin.org/articles/10.3389/fmicb.2023.1153153/full